The overarching goal of the work proposed is to understand the mechanisms of kidney development and regeneration with the long-term goal of establishing therapies for the treatment of acute and chronic kidney disease. Specifically, we will develop strategies to differentiate human inducible pluripotent stem (iPS) cells into proximal tubular epithelial cells. In our first specific aim, the goal is to understand gene expression profiles important to proximal tubule formation. We will approach this using two different experimental systems: 1) normal differentiation of proximal tubules during embryonic development, and 2) regeneration of proximal tubules following injury to the adult kidney. In both of these systems, proximal tubular cells will be isolated at key time points in each of these processes using laser capture microdissection (LCM). Using the collected tissue samples, gene expression profiling will be carried out using gene microarrays and real-time PCR. These experiments will allow us to select candidate genes that are important to inducing or regulating the proximal tubular cell fate. In the second specific aim, we will determine a set of conditions by which human iPS cells can be differentiated into proximal tubular cells. We will investigate the ability of our selected candidate genes and other genes known to be critical for normal kidney development to direct iPS cells toward the proximal tubular cell fate. We will also investigate the role of exogenous growth factors and extracellular matrix preparations in promoting the proximal tubular cell fate. Populations of human iPS cell-derived proximal tubular cells would be novel and important for incorporation into 3D structures to construct kidney tubules, as a first step to bioengineering patient-specific kidney tissue;incorporation into renal assist devices to treat patients with severe forms of kidney injury;development of in vitro models of patient-specific kidney diseases;and the development of kidney-specific toxicity screens for pharmaceuticals. Relevance: Chronic kidney disease (CKD), the gradual and usually permanent loss of kidney function over time, is a significant worldwide public health problem. Although recent advances in stem cell research have given hope to the use of human stem cells in the treatment of many human diseases, more research is needed to understand how stem cells can be used to treat kidney disease. The purpose of this study is to develop strategies for transforming stem cells into functioning kidney cells, with the ultimate goal of regenerating kidney tissue to treat patients with CKD.